Reduced flouride crustacean de-oiled protein-phospholipid complex compositions

ABSTRACT

The present invention contemplates the creation of a low fluoride oil processed from a phospholipid-protein complex (PPC) formed immediately upon a crustacean (i.e., for example, krill) catch. The process comprises disintegrating the crustaceans into smaller particles, adding water, heating the result, adding enzyme(s) to hydrolyze the disintegrated material, deactivating the enzyme(s), removing solids from the enzymatically processed material to reduce fluoride content of the material, separating and drying the PPC material. Then, using extraction with supercritical CO 2  and ethanol as solvents, inter alia krill oil is separated from the PPC. In the extraction the krill oil can be separated almost wholly from the feed material. The products have low fluoride content. The manufacturing costs in the extraction process are relatively low.

RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 13/856,642filed on Apr. 4, 2013, which is a Divisional of application Ser. No.13/342,664 filed on Jan. 3, 2012 (now issued as U.S. Pat. No. 8,557,297)which is a continuation-in-part of application Ser. No. 13/063,488 filedon May 24, 2011 (now issued as U.S. Pat. No. 8,758,829) which claimspriority to PCT/NO2009/000322 filed on Sep. 14, 2009, which has priorityto NO 20083906 filed on Sep. 12, 2008.

FIELD OF THE INVENTION

The invention relates to a method for processing crustaceans (i.e., forexample, krill) rich in lipids to produce low fluoride compositionscomprising phospholipids, proteinaceous nutrients and oil (i.e., forexample, neutral lipids and/or triglycerides).

BACKGROUND OF THE INVENTION

The crustaceans, especially krill, represent a vast resource asbiological material. The amount of Antarctic krill (Euphausia superba),depending on the calculation method and investigation, is roughly 1 to2×10⁹ tons and the possible weight of the annual catch is estimated at 5to 7×10⁶ tons. These small crustaceans, which live in the cold watersaround the Antarctic, are interesting as a source for proteins, lipidssuch as phospholipids, poly-unsaturated fatty acids etc.,chitin/chitosan, astaxanthin and other carotenoids, enzymes and othermaterials.

Several methods for isolating above-mentioned materials have beendeveloped. One problem is that the products may contain unwanted tracematerial included in the exoskeleton (also called integument or cuticle)of the crustaceans. For example, krill accumulates fluoride in theirexoskeleton, thereby increasing the fluoride amount of any producedmaterial either through the inclusion of parts of the exoskeleton orthrough extraction processes not taking into account the transfer offluoride to the final material. In this case free fluoride or looselybound fluoride may diffuse from the exoskeletal material and into thefurther processed material, making the end product high in fluoride ionsand/or fluorinated compounds.

Fluoride is a compound that in high concentrations is detrimental forthe health of land-dwelling animals as well as all kind of fish andcrustaceans and especially fresh-water fish species, since fluorideatoms have the tendency of entering into the bone structure of suchorganisms and creating fluorosis, or weakening of the bone structuresimilar in its effect to osteoporosis, but different since it is thebone structure itself, and not the porosity of the bone that isaffected. Skeletal fluorosis is a condition characterized by skeletalabnormalities and joint pain. It is caused by pathological boneformation due to the mitogenic action of fluoride on osteoblasts. In itsmore severe forms, skeletal fluorosis causes kyphosis, crippling andinvalidism. Secondary neurological complications in the form ofmyelopathy, with or without radiculopathy, may also occur. High fluorideintake has also been shown to be toxic to the male reproductive systemin rat experiments, and in humans high fluoride intake and symptoms ofskeletal fluorosis have been associated with decreased serumtestosterone levels. Consequently, if krill material is used as astarting material for food or feed products, precautions have to betaken for removing fluoride through the processing steps. However, thediffusion of fluoride and the presence of miniscule particles of theexoskeleton represent a problem that is difficult to overcome whenprocessing krill material in an industrial scale.

Polar lipids such as phospholipids are essential for cell membranes andare also called membrane lipids. For most known animal species thecontent of polar lipids is nearly constant. However, this does not holdfor the Antarctic krill. The phospholipids content varies from 2% up to10% depending on the season. The high content, e.g. more than 5%, of thephospholipids is in principle good, but means also a problem, because itmay result in strong emulsions in industrial processes. The emulsionscomplicate the separation of the lipid and proteinaceous fractions inthe processes, such as hydrolysis.

The krill oil is one the valuable products made from krills. It containsinter glia phospholipids, triglycerides and carotenoid astaxanthin whilebeing essentially free of protein, carbohydrates and minerals. Differentportions of the krill material are separated from each other by, interalia: i) crushing krill mechanically; ii) pressing them, iii) hydrolysiswith heat and enzymes; iv) centrifugal force in rotating devices; and v)solvent extraction.

What is needed in the art are significant improvements to these ratherconventional approaches and are described within many embodiments of thepresent invention (infra). For example, a disintegrated raw crustaceanmaterial may be separated and/or extracted into various enrichedlow-fluoride crustacean meal and/or oil compositions.

SUMMARY

The invention relates to a method for processing crustaceans (i.e., forexample, krill) rich in lipids to produce low fluoride compositionscomprising phospholipids, proteinaceous nutrients and oil (i.e., forexample, neutral lipids and/or triglycerides).

In one embodiment, the present invention contemplates aphospholipid-peptide complex (PPC) composition comprising a rangebetween approximately 40-50% lipids and less than 0.5 mg/Kg fluoride. Inone embodiment, the lipids comprise phospholipids.

In one embodiment, the present invention contemplates an oil compositioncomprising approximately 400-500 grams/Kg phospholipids, approximately200-260 grams/Kg Omega-3 fatty acids, less than 0.5 mg/Kg fluoride,approximately 15 grams/Kg lysophosphatidic acid, and less thanapproximately 8 grams/Kg free fatty acids.

In one embodiment, the present invention contemplates a de-oiledphospholipid-peptide complex (PPC) composition comprising approximately300-400 grams/Kg lipids, wherein approximately 0.1-1.0% are free fattyacids and a range between approximately 22-27% (w/w) that are Omega-3fatty acids. In one embodiment, the lipids comprise phospholipids.

In one embodiment, the present invention contemplates a crustacean lipidcomposition comprising at least 75% phospholipids. In one embodiment,the lipid composition comprises between approximately 75%-90%phospholipids. In one embodiment, the lipid composition comprisesbetween approximately 75%-80% phospholipids.

In one embodiment, the present invention contemplates a dried proteinhydrolysate composition comprising approximately 70-80% protein,approximately 1.5-3.0% lipids, and approximately 5-7% ash.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a hydrolyzed and disintegrated crustaceanmaterial; ii) at least one horizontal centrifuge capable of separatingsaid hydrolyzed crustacean material; and iii) a fluid comprising anon-polar solvent; and b) separating said hydrolyzed crustacean materialinto a high fluoride solid fraction and a low fluoride hydrolyzedmaterial fraction with a first horizontal centrifuge; c) separating saidlow fluoride hydrolyzed material fraction into a phospholipid-peptidecomplex (PPC) composition subfraction and a concentrated hydrolysatesubfraction with a second horizontal centrifuge; and d) contacting saidPPC composition subfraction with said non-polar solvent, wherein a lowfluoride oil is extracted. In one embodiment, the disintegratedcrustacean material has particle sizes between approximately 1-25millimeters. In one embodiment, the first horizontal centrifuge avoidsemulsification of said hydrolyzed crustacean material. In oneembodiment, the non-polar solvent comprises supercritical CO₂. In oneembodiment, the contacting further comprises a non-polar solvent. In oneembodiment, the non-polar solvent is ethanol. In one embodiment, thesecond horizontal centrifuge comprises an extended separation pathway.In one embodiment, the contacting is performed at a pressure of lessthan 300 bar. In one embodiment, the non-polar solvent further extractsa de-oiled PPC composition from said PPC composition subfraction. In oneembodiment, the ethanol separates a phospholipid composition and aprotein hydrolysate composition from said de-oiled PPC composition. Inone embodiment, the de-oiled PPC is separated from the PPC in less thanten hours. In one embodiment, the de-oiled PPC is separated from the PPCin less than five hours. In one embodiment, the de-oiled PPC isseparated from the PPC in less than two hours. In one embodiment, thehydrolyzed crustacean material comprises hydrolyzed krill material. Inone embodiment, the separating said hydrolyzed crustacean material isperformed at a centrifugal force of between approximately 1,000-1,800 g.In one embodiment, the separating said low fluoride hydrolyzed materialfraction is performed at a centrifugal force of between approximately5,000-10,000 g. In one embodiment, the method produces aphospholipid-peptide complex (PPC) composition comprising a rangebetween approximately 40%-50% lipid and less than 0.5 mg/Kg fluoride. Inone embodiment, the method produces an oil composition comprisingapproximately 400-500 grams/Kg phospholipids, approximately 200-260grams/Kg Omega-3 fatty acids, less than 0.5 mg/Kg fluoride,approximately 15 grams/Kg lysophosphatidic acid, and less thanapproximately 8 grams/Kg free fatty acids. In one embodiment, the methodproduces a de-oiled phospholipid-peptide complex (PPC) compositioncomprising approximately 300-400 grams/Kg lipids, wherein approximately0.1-1.0% are free fatty acids and a range between approximately 20-28%(w/w) are Omega-3 fatty acids. In one embodiment, the method produces acrustacean lipid composition comprising at least 75% phospholipids. Inone embodiment, the lipid composition comprises a range betweenapproximately 75%-90% phospholipids. In one embodiment, the lipidcomposition comprises a range between approximately 75%-80%phospholipids. In one embodiment, the method produces a dried proteinhydrolysate composition comprising approximately 70-80% protein,approximately 1.5-3.0% lipids, and approximately 5-7% ash.

In one embodiment, the present invention contemplates a systemcomprising: a) a solvent unit comprising at least non-polar solventinlet; b) an extraction tank unit in fluidic communication with thesolvent unit, wherein the tank comprises an inlet configured to receivea phospholipid-protein complex composition; c) a separator unitcomprising an outlet configured to release a low fluoride oilcomposition and residual co-solvent, wherein the separator is in fluidiccommunication with the tank; d) an absorbent unit in fluidiccommunication with the separator unit, wherein the absorbent unit iscapable of recycling the non-polar solvent. In one embodiment, thenon-polar solvent is a supercritical fluid. In one embodiment, thesupercritical fluid comprises carbon dioxide. In one embodiment, thesolvent unit further comprises a co-solvent inlet. In one embodiment,the co-solvent is a polar solvent. In one embodiment, the polar solventis ethanol. In one embodiment, the at least one non-polar solvent inletcomprises an unused non-polar solvent inlet. In one embodiment, the atleast one non-polar solvent inlet comprises a recycled non-polar solventinlet. In one embodiment, the solvent unit further comprises a fluidpump. In one embodiment, the tank unit is pressurized by the fluid pump.In one embodiment, the solvent unit further comprises a heater. In oneembodiment, the phospholipid-protein complex composition in the tankunit is heated by the heater. In one embodiment, the separator outlet isin fluid communication with an evaporator. In one embodiment, theseparator further comprises a horizontal centrifuge. In one embodiment,the horizontal centrifuge is a decanter centrifuge having an extendedseparation pathway. In one embodiment, the phospholipid-protein complexcomposition is a low fluoride crustacean phospholipid-protein complexcomposition. In one embodiment, the low fluoride crustaceanphospholipid-protein complex composition is a low fluoride krillphospholipid-protein complex composition.

In one embodiment, the present invention contemplates a method forprocessing crustaceans, especially krills, in which method thecrustaceans are disintegrated into smaller particles, fresh water isadded to the disintegrated material, the water with the disintegratedmaterial is heated and enzyme(s) are added for hydrolyzing thedisintegrated material and said enzyme(s) is/are deactivated, the methodfurther comprising steps: a) removing solids from the hydrolyzedmaterial to reduce fluoride content of the material; b) separatingphospholipid-peptide complex material and concentrated hydrolysatefraction from each other; c) drying said phospholipid-peptide complexmaterial; and d) dividing the drying result, or PPC, to components byextraction(s) using at least a supercritical CO₂ as solvent, wherein theprocessing of crustaceans is started as soon as a crustacean catch hasbeen raised on ship. In one embodiment, the fluoride content solids areremoved from the hydrolyzed material by a decanter. In one embodiment,the phospholipid-peptide complex material and concentrated hydrolysatefraction are separated from each other by a sedicanter with highcentrifugal forces and long clarification/separation zones to avoid anemulsification. In one embodiment, the method further comprises using inthe extraction ethanol as a co-solvent in addition to the supercriticalCO₂ to separate: i) a krill oil consisting of phospholipids andtriglycerides, or neutral oil, and ii) a protein hydrolysate from thePPC. In one embodiment, the pressure of the solvent being at most 300bar. In one embodiment, the extraction includes two steps: i) firstusing only the supercritical CO₂ as solvent to separate de-oiled PPCfrom the PPC; and ii) second using only ethanol as solvent to separatephospholipids and protein hydrolysate from the de-oiled PPC. In oneembodiment, the duration of the step when said de-oiled PPC is extractedfrom the PPC is at most three hours. In one embodiment, the methodproduces a phospholipid-peptide complex (PPC) composition comprisingapproximately 40%-50% lipid and approximately 0.5 mg/kg fluoride. In oneembodiment, the lipid comprises phospholipids. In one embodiment, themethod produces an oil composition comprising approximately 400-500grams/Kg phospholipids, approximately 200-260 grams/Kg Omega-3 fattyacids, approximately 0.5 mg/Kg fluoride, approximately 15 grams/Kglysophosphatidic acid, and less than approximately 8 grams/Kg free fattyacids. In one embodiment, the method produces a de-oiledphospholipid-peptide complex (PPC) composition comprising approximately300-400 grams/Kg lipids, wherein approximately 0.1-1.0% are free fattyacids and approximately 22-27% (w/w) are Omega-3 fatty acids. In oneembodiment, the method produces a crustacean phospholipid compositioncomprising approximately 75% polar lipids. In one embodiment, the methodproduces a dried protein hydrolysate composition comprisingapproximately 70-80% protein, approximately 1.5-3.0% lipids, andapproximately 5-7% ash.

DEFINITIONS

The term “disintegrated material” as used herein refers to anybiological material that has been subjected to a mechanical destructionand/or disruption that results in a composition having particle sizes ofbetween approximately 1-25 millimeters, preferably between approximately3-15 millimeters, more preferably between approximately 5-10 millimetersand most preferably approximately 8 millimeters.

The term “hydrolyzed material” as used herein refers to any biologicalmaterial that has been subjected to high heat and/or enzymatictreatment. Such hydrolyzed materials would be expected to havephospholipid/peptide components that are physically separated from thecomponents of the chitinous exoskeleton.

The term “crustacean” as used herein refers to any marine organism havea hard outside shell (e.g., a chitinous exoskeleton combined with acarbonate) encompassing a fleshy interior that is a living organism.More specifically, the crustaceans are usually considered a large classof mostly aquatic arthropods that have a chitinous or calcareous andchitinous exoskeleton, a pair of often much modified appendages on eachsegment, and two pairs of antennae. For example, a crustacean mayinclude but not limited to, krill, lobsters, shrimps, crabs, wood lice,water fleas, and/or barnacles.

The term “horizontal centrifuge” refers to any device that is capable ofrotating a mixture in the Z-plane (as opposed to the X-plane and/orY-plane as with conventional centrifuges). This rotation is generated bya screw-type conveyor element aligned horizontally within a tube shapedenclosure. The induced centrifugal force then layers the heavierparticles to the outside edges of the enclosure, while the lighterparticles form layers closer to the center of the enclosure. Somehorizontal centrifuges are modified to comprise an extended separationpathway and induce high gravitational forces (e.g., a sedicanter).

The term “polar solvent” as used herein refers to any compound, orcompound mixture, that is miscible with water. Such polar solventcompounds include, but are not limited to, ethanol, propanol and/orethyl acetate.

The term “non-polar solvent” as used herein refers to any compound, orcompound mixture, that is not miscible with water. Such non-polarsolvent compounds include, but are not limited to, hexane, pentaneand/or supercritical CO₂.

The term “supercritical CO₂” refers to any mixture comprising carbondioxide (CO₂) in a fluid state while held at, or above, its criticaltemperature and critical pressure where its characteristics expand tofill a container like a gas but with a density like that of a liquid.More specifically, carbon dioxide becomes a supercritical fluid above31.1° C. and 72.9 atm/7.39 MPa. Carbon dioxide usually behaves as a gasin air at standard temperature and pressure (STP), or as a solid calleddry ice when frozen. If the temperature and pressure are both increasedfrom STP to be at or above the critical point for carbon dioxide, it canadopt properties midway between a gas and a liquid. As contemplatedherein, supercritical CO₂ can be used as a commercial and industrialsolvent during chemical extractions, in addition to its low toxicity andminimal environmental impact. The relatively low temperature of theprocess and the stability of CO₂ also allows most compounds (i.e., forexample, biological compounds) to be extracted with little damage ordenaturing. In addition, because the solubility of many extractedcompounds in CO₂ may vary with pressure, supercritical CO₂ is useful inperforming selective extractions.

The term “fluoride” as used herein interchangeably and refer to anycompound containing an organofluoride and/or an inorganic fluoride.

The term “high fluoride solid fraction” as used herein refers to acomposition containing the vast majority of a crustacean's exoskeletonfollowing a low g-force (e.g., between approximately 1,000-1,800 g)horizontal centrifugation separation of a hydrolyzed and disintegratedcrustacean material. This fraction contains small particles ofexoskeleton of the crustacean that retains the vast majority of fluoride(i.e., for example, between 50-95%) in these organisms.

The term “low fluoride” as used herein may refer to the product of anymethod and/or process that reduced the fluoride from the originalmaterial by approximately 10-fold (i.e., for example, from 5 ppm to 0.5ppm). For example, ‘a low fluoride crustacean phospholipid-proteincomplex’ comprises ten-fold less fluoride than ‘a low fluoridehydrolyzed and disintegrated crustacean material’.

The term “low fluoride hydrolyzed material fraction” as used hereinrefers to a composition containing the vast majority of a crustacean'sfleshy internal material following a low g-force (e.g., betweenapproximately 1,000-1,800 g) horizontal centrifugation separation of ahydrolyzed and disintegrated crustacean material. This fraction containssmall particles of phospholipids, neutral lipids, proteins and/orpeptides that is largely devoid of any fluoride (i.e., for example,between 5%-50% of the raw hydrolyzed and disintegrated material).

The term “a low fluoride phospholipid-peptide complex compositionsubfraction” as used herein refers to a low fluoride compositioncontaining the vast majority of lipid material following a high g-force(e.g., between approximately 5,000-10,000 g) horizontal centrifugationseparation of a low fluoride hydrolyzed material fraction.

The term “concentrated hydrolysate composition subfraction” as usedherein refers to a low fluoride composition containing the vast majorityof water soluble lean material following a high g-force (e.g., betweenapproximately 5,000-10,000 g) horizontal centrifuge separation of a lowfluoride hydrolyzed material fraction.

The term “low fluoride oil” as used herein refers to a lipid-richcomposition created by the extraction of a phospholipid-peptide complexcomposition subfraction using a selective extraction process, such aswith a supercritical carbon dioxide fluid. Such a process removesapproximately ten-fold of the fluoride from the raw hydrolyzed anddisintegrated crustacean material.

The term “de-oiled phospholipid-peptide complex” as used herein refersto a low fluoride composition containing the vast majority of dry mattercomposition created by the extraction of a phospholipid-peptide complexcomposition subfraction using selective extraction process, such as asupercritical carbon dioxide fluid.

The term “phospholipid composition” as used herein refers to a lowfluoride composition comprising a high percentage of polar lipids (e.g.,approximately 75%) created by the extraction of a de-oiledphospholipid-peptide complex using a co-solvent, such as ethanol.

The term “protein hydrolysate” as used herein refers to a low fluoridecomposition comprising a high percentage of protein (e.g., approximately70-80%) created by the extraction of a de-oiled phospholipid-peptidecomplex using a co-solvent, such as ethanol.

The term “immediately” as used herein refers to a minimum practicalperiod between decking a crustacean catch in a trawl bag and/or netcoupled with a direct transfer to a suitable disintegraor. For example,this minimum practical period should preferably not exceed 60 minutes,more preferred to not exceed 30 minutes, even more preferred to notexceed 15 minutes.

The term “hydrolysis” as used herein refers to any break and/ordisruption made in a protein structure of a disintegrated crustaceanmaterial, wherein in the naturally occurring protein sequences becomeshorter (i.e., for example, by breaking peptide bonds of the amino acidsequence primary structure) and/or denatured (i.e., for example, anunfolding of the amino acid sequence secondary, tertiary and/orquaternary structure). This process may be controlled by hydrolyticenzyme(s). For example, one or more exogenous proteolytic enzymes (e.g.alkalase, neutrase, and enzymes derived from microorganisms or plantspecies) may be used in the process. Co-factors such as specific ionscan be added depending on the used enzymes. The selected enzyme(s) canalso be chosen for reducing emulsions caused by high content ofphospholipids in the raw material. Besides the temperature, thehydrolysis takes place within optimal or near-optimal pH and sufficienttime. For example, the exogenous enzyme alkalase the optimum pH is about8, optimum temperature about 60° C. and the hydrolysis time 40-120minutes.

The term “solvent unit” refers to any enclosed volume configure to heatand pressurize a mixture of supercritical carbon dioxide fluid and/or aco-solvent (e.g., ethanol). Such an enclosed volume may be constructedout of any suitable material including but not limited to metals (e.g.,steel, aluminum, iron etc.), plastics (e.g., polycarbonate, polyethyleneetc.), fiberglass (etc.).

The term “extraction tank” refers to any enclosed volume configured towithstand heat and pressure sufficient to perform lipid and proteinextraction from a raw biomass using a supercritical carbon dioxidefluid. As designed, the extraction tank contemplated herein isconfigured such that the solvents containing the extracted lipids andproteins rise to the tank top for transfer to a separator unit. Such anenclosed volume may be constructed out of any suitable materialincluding but not limited to metals (e.g., steel, aluminum, iron etc.),plastics (e.g., polycarbonate, polyethylene etc.), fiberglass (etc.).

The term “separator unit” refers to any enclosed volume configured witha centrifuge capable of separating the components of the extractedlipids and proteins received from an extraction tank. The respectiveextraction components exit the separator unit via outlet ports such thatthe remaining solvents (i.e., supercritical CO₂) are transferred to anabsorbent unit for recycling. Such an enclosed volume may be constructedout of any suitable material including but not limited to metals (e.g.,steel, aluminum, iron etc.), plastics (e.g., polycarbonate, polyethyleneetc.), fiberglass (etc.).

The term “absorbent unit” refers to any enclosed volume configured withmaterials that will remove contaminants from a supercritical CO₂ fluid.Such materials may include, but are not limited to charchol, coal,purifying gases, plastic polymer resins and/or filtration cartridgescomprising single or dual-flat extruded nets (Tenax UK LTD, Wrexham,North Wales LL13 9JT, UK). Such an enclosed volume may be constructedout of any suitable material including but not limited to metals (e.g.,steel, aluminum, iron etc.), plastics (e.g., polycarbonate, polyethyleneetc.), fiberglass (etc.).

The term “in fluidic communication” refers to any means by which a fluidcan be transported from one location to another location. Such means mayinclude, but are not limited to pipes, buckets and/or troughs. Suchmeans may be constructed out of any suitable material including but notlimited to metals (e.g., steel, aluminum, iron etc.), plastics (e.g.,polycarbonate, polyethylene etc.), fiberglass (etc.).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents a flow diagram of one embodiment of a method to producea low fluoride crustacean material.

FIG. 2 presents a longitudinal centrifuge with an extended separationpath. This specific example is a FLOTTWEG SEDICANTER horizontal decantercentrifuge.

FIG. 3 depicts one example of an extraction plant suitable for use inthe presently disclosed method. For example, the plant comprises asolvent unit (21), an extraction tank (22), separators (23) andadsorbents (24).

FIG. 4 present exemplary data showing the extraction efficiencies of twodifferent runs in accordance with one embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method for processing crustaceans (i.e., forexample, krill) rich in lipids to produce low fluoride compositionscomprising phospholipids, proteinaceous nutrients and oil (i.e., forexample, neutral lipids and/or triglycerides).

Krill oil comprises lipids extracted with solvents from krill biomass.Krill biomass can be either fresh, whole krill (WO2008/060163A1), frozenwhole krill (Neptune Technologies & Bioresources Inc., Canada),lyophilized whole krill (JP2215351) or krill meal (US20080274203).Solvents used in extracting lipids from krill biomass have been reportedas acetone+ethanol (WO2000/23546; WO2002/102394), ethanol+hexane(Enzymotec Ltd), ethanol alone (JP2215351; Aker BioMarine ASA, Norway)or supercritical CO₂+ethanol co-solvent (US2008/0274203; WO2008/060163).Solvent-free technology for obtaining krill oil has also been developed(US20110224450A1). Krill oil comprises a lipid fraction of raw krillbiomass that is essentially free of protein, carbohydrates and/orminerals. Krill oil also comprises neutral lipids (e.g., mostlytriglycerides), polar lipids (e.g., mostly phospholipids) and carotenoidastaxanthin. Although it is not necessary to understand the mechanism ofan invention, it is believed that the lipid and/or fatty acidcompositions of krill oil vary depending of the season.Phosphatidylcholine is the largest phospholipid component of krill oiland its proportion is relatively stable.

In some embodiments, the present invention contemplates methods ofprocessing crustacean biomass having unexpected findings including, butnot limited to: i) removal of most of the exoskeleton from thecrustacean biomass that results in low level of fluorides in PPC andvery low levels of fluoride in krill oil extracted from PPC bysupercritical CO2 and ethanol co-solvent; ii) a level of fluorides inthe crustacean oil that is less than 0.5 ppm in contrast to conventionalkrill oil with fluoride content of greater than 0.5 to 5 ppm; iii)crustacean oil extracted from PPC by supercritical CO₂ and ethanolco-solvent has a minimal brown color suggesting that minimal degradationof astaxanthin or formation of tertiary oxidation products has occurred;iv) it is believed that the level of dark/brown color measured on HunterL*a b scale and the pyrrole content measured by absorption at 570 nm arelower in the low fluoride, supercritical CO₂ and ethanol co-solventextracted crustacean oil as compared to the conventional krill oil; v)analysis of the low fluoride, supercritical CO₂ and ethanol co-solventextracted crustacean oil indicates a minimal content of FFA and LPC.These findings suggest that the lipids of crustacean biomass haveundergone minimal hydrolysis during the initial processing stepsproducing PPC; and vi) levels of FFA in the low fluoride, supercriticalCO₂ and ethanol co-solvent crustacean oil are less than 0.8 g/100 oiland LPC content less than 1.5 g/100 g of oil.

I. Historical Overview of Crustacean Processing Methods

Publication GB 2240786 discloses a method for processing krill includingremoving a part of the fluoride content of krill. The removing is basedon passing electric current through pulverized krill. However,fluoride-containing solid particles remain in the material.

Publication US 2011/0224450 (Sclabos Katevas et al., herein incorporatedby reference) discloses a method for obtaining krill oil from whole rawkrills using inter alia cooking, separating by decanter, and pressing.No solvents and extraction are used.

Publication WO 2008/060163 (Pronova Biopharma AS) discloses a method forobtaining krill oil using supercritical CO₂ and either ethanol,methanol, propanol or isopropanol as co-solvent. Fresh or pre-heated(about 90° C.) whole krills are used as the extraction feed material.

Publication WO 02/102394 (Neptune Technologies & Bioresources) disclosesa method for obtaining krill oil using in different phases acetone andethanol or e.g. ethyl acetate as solvents. Frozen whole krill is used asfeed material.

Publication JP 2215351 (Taiyo Fishery) discloses a method for obtainingkrill oil using ethanol as solvent. Lyophilized whole krills are used asfeed material.

Publication US 2008/0274203 (Aker Biomarine ASA, Bruheim et al.)(hereinincorporated by reference) discloses a method for obtaining krill oilfrom krill meal using supercritical fluid extraction in a two-stageprocess. Stage 1 removes the neutral lipid by extracting with neatsupercritical CO₂ or CO₂ plus approximately 5% of a co-solvent. Stage 2extracts the actual krill oils using supercritical CO₂ in combinationwith approximately 20% ethanol.

There are a number of problems associated with these conventionallyknown technologies of extracting krill lipids, including but not limitedto: i) whole crustacean biomass contains high fluoride exoskeletonparticles that results in the production of fluoride-contaminatedcrustacean oil; ii) crustacean oil having a brownish hue color may arisefrom exposing astaxanthin to excessive heat during crustacean biomassprocessing. Specifically, brown color can arise from degradation ofastaxanthin and/or from accumulation of the end products ofnon-enzymatic browning (e.g., Strecker degradation products orpolymerized pyrroles). Brown color resulting from this non-enzymaticprocess is believed to result from oxidative degradation due to areaction of secondary lipid oxidation products with amino groups fromamino acids or proteins creating so-called tertiary oxidation products;iii) freezing the crustacean biomass for transportation to an extractionplant. Frozen crustacean biomass can be relatively stable, but somechanges in the product are known to occur over time. For example, onecharacteristic change in frozen krill is partial hydrolysis of thelipids resulting in accumulation of free fatty acids (FFA) arising fromboth triglycerides and phospholipids and lysophospholipids, specificallylysophophatidylcholine (LPC), arising from hydrolysis ofphosphatidylcholine; iv) the use of heat and frozen storage can induceoxidation of lipids and proteins in crustacean biomass. Primaryoxidation leads into formation of secondary oxidation products that arevolatile and can be detected in krill oil as off-flavors or undesirableodor; and v) the separation of the krill oil from the feed material isquite inefficient, wherein only about a half of the oil can beextracted.

II. Production of Low Fluoride Crustacean Materials

In one embodiment, the present invention contemplates a methodcomprising forming a phospholipid-peptide complex (PPC) composition froma crustacean (i.e., for example, krill) immediately after the catch hasbeen brought upon on board a boat and/or ship (i.e., for example, afishing vessel). The process of creating the PPC composition comprisesdisintegrating the crustaceans into a disintegrated material comprisingsmaller particles (i.e., for example, between approximately 1-25millimeters), adding water, heating the disintegrated material, addingenzyme(s) to hydrolyze the disintegrated material, deactivating theenzyme(s), removing solids (i.e., for example, exoskeleton, shell,and/or carapace) from the enzymatically processed material to reduce thefluoride content of the material, separating and drying the PPCcomposition. Preferably, the PPC composition is transferred to anon-shore facility (i.e., a fish oil extraction plant) where alow-fluoride crustacean oil is separated from the PPC composition usingsolvents including, but not limited to, supercritical CO₂ and/orethanol. Using alternative extractions, de-oiled PPC compositions,phospolipids and/or protein hydrolysate compositions are also separatedfrom the PPC composition.

-   -   An advantage of some embodiments of the invention is that these        crustacean products, like krill oil, have a low fluoride        content. This is due to the fact that the solid crusteacean        exoskeletal particles (i.e., for example, shell and/or carapace)        are effectively removed from mass to be processed.    -   Another advantage of the invention is that crustacean oil can be        separated effectively, almost completely, from the disintegrated        crustacean material (e.g., feed material) during the extraction.        This is due to the fact that, in the extraction process with the        supercritical CO₂ solvent, the feed material comprises a PPC        composition. Although it is not necessary to understand the        mechanism of an invention, it is believed that the phospholipids        of the feed material are embedded in a matrix of hydrolyzed        protein which means that the close association between the        phospholipids and hydrophobic/phosphorylated proteins is broken        thus facilitating the extraction of the lipids.    -   An advantage of the invention is that relatively low pressure        and temperature can be used in the extraction, which means lower        production costs.    -   A further advantage of the invention is that disposal of        residual solvents, common when using other more conventional        lipid solvents, is avoided when using supercritical CO₂ as a        solvent.    -   A further advantage of the invention is that also the        phosphatidylserine (PS), free fatty acids (FFA) and        lysophosphocholine (LPC) contents are very low in the end        products.    -   A further advantage of the invention is that a low fluoride        crustacean oil product (i.e., for example, a low fluoride krill        oil) has very little brown color. It is believed in the art that        apperance of a brown color in crustacean oil indicates that        unfavorable processes are occuring during the manufacture of the        feed material (e.g., a disintegrated crustacean material).

A. Processing Of Crustaceans

The present invention provides an industrial method for processingcatches of crustaceans comprising a number of steps beginning with avery early and substantially complete removal of the crustacean'sexoskeleton (i.e., for example, the crust, carapace and/or shell).Although it is not necessary to understand the mechanism of aninvention, it is believed that the crustacean exoskeleton comprises avast majority of fluoride in the organism. Consequently, this stepthereby results in a substantial removal of fluoride from the crustaceanmaterial. The method also uses longitudinal centrifugation techniquesthat prevents separation problems caused by emulsions when processing araw material with high content of phospholipids.

The method according to the present invention is initiated immediatelyafter decking a catch of crustacean. It is of importance that the methodaccording to the present invention is initiated as soon as possibleafter the crustacean catch has been decked since fluoride starts toleak/diffuse immediately from the exoskeleton into the crustacean'sflesh and juices.

When using the term “immediately” in connection with starting theprocess according to the present invention this relates to the periodfrom decking the crustacean catch and to the initial disintegration ofthe crustacean (see infra). This period of time should be kept to aminimum, and should preferably not exceed 60 minutes, more preferred notexceed 30 minutes, even more preferred not exceed 15 minutes, and shouldinclude a direct transfer of the krill catch from the trawl bag and/ornet to a suitable disintegrator. A disintegrator of the crustaceanmaterial may be a conventional pulping, milling, grinding or shreddingmachine.

The crustacean catch is initially loaded into a disintegration appratuswhere the crustacean catch is subjected to pulping, milling, grindingand/or shredding to create a disintegrated crustacean material. Thetemperature of the disintegration process is around the ambienttemperature of the water, i.e. between −2 and +1° C., preferably around+0° C. to +6° C., and may be performed by any convenient disintegrationmethod. This disintegration process is also conventionally done by theprevious known processing methods, and represents one of the obstaclesaccording to the prior art because it produces large amounts ofexoskeletal particles from the crustacean mixing in the milled materialand producing a disintegrated paste with a high fluoride content.However, this high fluoride content is one of the reasons why the priorart processed crustacean material has limited applications and is lesssuitable for food, feed or corresponding food or feed additives comparedto other marine raw materials e.g. pelagic fish.

According to the present invention the crustacean material is dividedinto a particle size suitable for a further separation step for notinterfering with the subsequent processing steps. The disintegratingprocess is performed continuously and produces particle sizes up to 25mm, a preferred particle size range is between approximately 0.5-10 mmand a more preferred size range is between approximately 1.0-8 mm.

Although it is not necessary to understand the mechanism of aninvention, it is believed that this small particle size distributionrepresents one of advantages of the present invention because thefluoride has a tendency to leak out of the milled material and minglewith the rest of the raw material. However, this leaking process takestime and is not rapid enough to negatively impact a subsequent enzymatichydrolysis step, provided the hydrolysis step is performed withinspecific parameters with respect to time and optimal, or near-optimalconditions, such as pH and temperature and optionally with the additionof co-factors such as specific ions depending on the used enzymes.

The temperature of the disintegrated material may, according to thepresent invention, be elevated to a temperature suitable for thesubsequent enzymatic hydrolysis. Preferably, the temperature may beincreased within seconds (e.g. 1-300 seconds, more preferred 1-100seconds, even more preferred 1-60 seconds, most preferred 1-10 seconds)subsequent to the disintegrating step for reducing the processing timeand thereby preventing diffusion of fluoride and for preparing thematerial for the enzymatic hydrolysis.

According to the present invention enzymes may be added directly to thedisintegrated material or through the added water or both, before,during or after the disintegration process.

According to the present invention, exogenous proteolytic enzymes (e.g.,alkalase, neutrase, enzymes derived from microorganisms including, butnot limited to, Bacillus subtilis and/or Aspergillus niger, and/or orenzymes derived from plant species) may be added before, during or afterthe disintegration, and before, during or after the heating of thedisintegrated material. The added enzyme(s) may be in the form of onesingle enzyme or a mixture of enzymes. The conditions of the hydrolysisshould match the optimal hydrolytic conditions of the added enzyme(s)and the selection of optimal conditions for the selected exogenoushydrolytic enzyme(s) is known to the person skilled in the art. As anexample, the exogenous enzyme alkalase having a pH optimum of about 8, atemperature optimum of 60° C. and a hydrolysis time of 40-120 minutes.The selected enzymes, or combination of enzymes, should also be chosenfor reducing emulsions caused by high content of phospholipids in theraw material.

An efficient amount of proteolytic enzyme(s) will be set after aprocess- and product optimization process that depends upon theefficiency of a specific chosen commercial enzyme or mix of enzymes. Atypical amount by weight of commercial enzymes, as a ratio of the amountof the weight of the disintegrated raw material, are preferably between0.5% and 0.05%, more preferably between 0.3% and 0.07% and mostpreferable between 0.2% and 0.09%. This hydrolysis step is aided byendogenous (natural) enzymes because rapid and uncontrolled autolysis iswell known in fresh caught crustaceans.

The reason for adding exogenous enzymes is to take control of and guide,the breakdown of the proteinaceous material in the disintegratedsubstance as well as speeding up/accelerating the hydrolysis of thematerial to avoid and/or preclude the leaking of fluoride from theshell, carapace and crust as mentioned supra. These hydrolytic enzymes,or a combination of hydrolytic enzymes, should also be carefully chosento reduce emulsion in the production process. Enzymes may be selectedfrom exo- and/or endopeptidases. If a mixture of enzymes is used, such amixture may also include one or more chitinases for subsequently makingthe chitin-containing fraction(s) more amenable to further downstreamprocessing. If chitinases are used, care must be taken for notincreasing the leakage of fluoride from the shell/crust/carapace of thecrustacean into the other fractions. However, since such fluorideleakage takes time, it is possible to perform such an enzymatictreatment within the time parameters indicated supra. A more convenientalternative to including chitinases in the enzyme mix of the initialhydrolysis step will be to process the separated chitin-containingfraction subsequently to the separation step.

As it is important to avoid the leaking of fluoride from the milledexoskeletal material into the milled fleshy material, and since theleaking to some degree is related to the increased surface area createdthrough the disintegrating step, the enzymatic hydrolysis step should befinished within a time interval of 100 minutes, preferably within 60minutes, most preferred within 45 minutes calculated from the additionof the endogenous enzyme(s). The amount of enzyme(s) added is related tothe type of enzyme product used. As an example it may be mentioned thatthe enzyme alkalase may be added in an amount of 0.1-0.5% (w/w) of theraw material. This should be taken into context with the addedendogenous enzymes since the addition of more enzymes will reduce thetime interval of the hydrolytic step. As mentioned supra the time of thehydrolytic step is one of the crucial features of the present processsince a short hydrolysis time reduces the diffusion time of fluoridefrom particles of the exoskeleton. The hydrolytic enzymatic processingstep is intended to remove the binding between the soft tissue of thekrill to the exoskeleton of the crustacean.

Subsequent to, or together with, the hydrolytic processing step thehydrolyzed and distintegraed crustacean material is passed through aparticle removal device operating through a gravitational force such asa longitudinal centrifuge (i.e., for example, a decanter). This firstseparation step removes the fine particles containing a considerableamount of the fluoride from the hydrolysed or hydrolysing crustaceanmaterial to create a solids fraction. The centrifuge is operated with ag force between 1,000 and 1,800 g, more preferably between 1,200 and1,600 g and most preferably between 1,300 and 1,500 g. Through thisparticle removal step a substantial amount of fluoride is removed fromthe proteinaceous crustacean fraction. The reduction of fluoride on adry weight basis as compared to conventional cnistacean meal, with atypical fluoride content of 1,500 p.p.m, may be up to 50%, even morepreferred up to 85%, most preferred up to 95%.

The enzymatic hydrolysis may be terminated by heating of the hydrolysingmaterial (incubate) to a temperature over 90° C., preferably between92-98° C. and most preferred between 92-95° C., prior to, during orafter the separation step, as long as the hydrolysis duration lieswithin the above given boundaries. The hydrolysis is terminated before,during, or after the fine particle removal step, most preferred afterthe fine particle removal step. The temperature of the firstcentrifugation particle removal step, in one embodiment, depend on theoptimal activity temperature of the enzyme (in the case where theenzymatic hydrolysis step is terminated by heating after the fineparticle separation step).

The fluoride content in the prior art processed krill protein materialhas limited applications and are less suitable for food or feed orcorresponding food or feed additives, as mentioned supra but thefluoride content of the removed exoskeletal material is not preventivefor further separation/purification of this fraction. Thus materialssuch as chitin, chitosan and astaxanthin may be isolated from theseparated exoskeletal material. Such isolation procedures are knownwithin the art. Steps may also be taken for removing the fluoride fromthe isolated exoskeletal material e.g. through dialysis, nanofiltration,through electrophoresis or other appropriate technologies.

Hydrolytic enzyme(s) deactivation may be performed in different ways,such as adding inhibitors, removing co-factors (e.g., crucial ionsthrough dialysis), through thermal inactivation and/or by any otherdeactivating means. Among these, thermal inactivation, as mentionedsupra, is preferred by heating the proteinaceous material to atemperature where the hydrolytic enzymes become denatured anddeactivated. However, if a product where the relevant native proteinsare not denatured is wanted, other means than heating for deactivatingthe hydrolytic enzymes should be selected.

A first centrifugation forms a de-fluorinated hydrolyzed anddisintegrated crustacean material fraction and a solids fraction (e.g.,containing high fluoride exoskeleton particles). As described below, thelow flourine hydrolyzed and disintegrated crustacean material fractionmay be subsequently separated (e.g., by a second centrifugation) to forma low fluoride Phospholipid-Peptide Complex (PPC) composition fraction,a lean low fluoride Concentrated Hydrolysate Fraction (CHF) fractionthat can be used as a food and/or feed additives, and a lipid fractionmainly consisting of neutral lipids. The PPC composition subfraction isrich in lipids, like a smooth cream with no particles, wherein thelipids are well suspended within the peptide components. This suspensionresults in small density differences between the different PPCcomposition components thereby making it difficult to further separatethe PPC composition with common centrifugal separators and/or decanters.This is especially accentuated with crustacean catches during the secondhalf of the fishing season.

Ordinary disc centrifugal separators (i.e., generating rotational forcein the X and Y plane) do not work properly to separate a PPC compositionsubfraction into its respective components since emptying and necessarycleaning cycles with water will disturb separation zones. Conventionalcentrifugation separation processes result in the formation of unwantedemulsion products having a high phospholipid content and low dry matterconcentrations. Standard decanters cannot separate the PPC compositionsubfraction into its respective components due to a low g forcelimitation, short separation zone and an intermixing of light and heavyphases at the discharge of heavy phase from the machine.

In one embodiment, the present invention contemplates a methodcomprising separating a low fluoride PPC material into subfractionsusing a horizontal decanter centrifuge with an extended separation path.See, FIG. 2. Horizontal centrifuges (e.g., generating a rotational forcein the Z plane) are useful for the present invention comprise modifiedconvention decanter centrifuges. For example, a PPC compositionsubfraction would enter an ordinary decanter from a bowl through acentral placed feed pipe in the middle of the separation zone. Incontrast, when using horizontal centrifuges as contemplated herein, thePPC composition subfraction enters at the end and at the opposite sideof the outlet (1). This modification provides a significant improvementin the separation process by providing a considerably longerclarification/separation zone than ordinary decanters and utilizes thetotal available separation length (2) of the machine. The drive is ableto impart high g-forces: 10,000 g for small machines and 5,000 to 6,000g for high capacity machines, facilitating the separation of very fine,slow-settling PPC composition subfractions without the complications ofemulsification. The PPC composition subfraction will be subjected to thehighest g-force just before entering under the baffle (3). The differentliquid layers separated from PPC composition subfraction areconcentrated gradually along the axis of the horizontal centrifugethereby exiting the machine under baffle (3) by the g force pressuregenerated by the machine (4). The separation of the PPC compositionsubfraction into a layer comprising about 27-30% dry matter makes thedownstream processing efficient in terms of operating/robustness and aswell economically considering both yield and costs of preparing the drymatter into a meal composition. The PPC composition subfractionseparation also creates a layer comprising a lean hydrolysate that canbe evaporated into a concentrated hydrolysate of greater than 60%.

B. Processing of Krill

One embodiment according to the invention is depicted as a flow diagramfor the processing of krill (steps 11 to 1A). See, FIG. 1. The functionaccording to the method, or the process according to the invention isinitiated as soon as a krill catch has been raised to the ship. Althoughit is not necessary to understand the mechanism of an invention, it isbelieved that fluoride immediately starts to leak/diffuse from thechitinous exoskeleton into the flesh and juices of the dead krills. ‘Assoon as’ means here a period at most 60 minutes, in practice, forexample 15 minutes. During this period the krill catch is transferredfrom the trawl/net to a suitable disintegrator. In the disintegrator thekrill material is crushed to relatively small particles. Thedisintegrating can be performed by any convenient method: pulping,milling, grinding or shredding. The temperature in the disintegrationprocess is around the ambient temperature of the water, i.e. between −2°C. and +10° C., preferably between +0° C. and +6° C. The disintegrationproduces large amount of chitinous debris among the rest of the krillmaterial, thereby contributing to a high fluoride content.

The particle size distribution of the disintegrated krill material issignificant because of the above-mentioned fluoride leak from thechitinous debris and to the rest of the raw material. It is believedthat the smaller particle sizes results in a more complete separation ofthe solids fraction from the disintegrated krill material (infra). Forthis reason the preferable range of the particle size is 1.0-8 mm.However, the leaking process is relatively slow and has not time to berealized during the following process phases.

Next, including in steps 11, fresh water is added to the disintegratedkrill material. The volume/L of the water added is, for example, same asthe weight/kg of the disintegrated krill material to be processed duringthe subsequent process phase of enzymatic hydrolysis. The temperature ofthe disintegrated krill material with the added water is increased suchthat it is suitable for the hydrolysis and enzyme(s) are added. Theheating is carried out fast, within at most five minutes, after thedisintegrating step to reduce the processing time and thereby to preventdiffusion of fluoride and to prepare the material for the enzymatichydrolysis. The enzyme(s) can be added directly to the disintegratedkrill material, or through the added water or both, before, during orafter the heating step.

The term “hydrolysis” as used herein, means that breaks are made in theprotein structure in the disintegrated substance, and the protein chainsbecome shorter. This process is controlled by hydrolytic enzyme(s). Forexample, one or more exogenous proteolytic enzymes (e.g. alkalase,neutrase, and enzymes derived from microorganisms or plant species) maybe used in the process. Co-factors such as specific ions can be addeddepending on the used enzymes. The selected enzyme(s) can also be chosenfor reducing emulsions caused by high content of phospholipids in theraw material. Besides the temperature, the hydrolysis takes place withinoptimal or near-optimal pH and sufficient time. E.g. for the exogenousenzyme alkalase the optimum pH is about 8, optimum temperature about 60°C. and the hydrolysis time 40-120 minutes.

The amount of proteolytic enzyme(s) can be set after a process- andproduct optimization, and depends naturally on the efficiency of thechosen enzyme or mix of enzymes. A typical ratio of the weight of addedcommercial enzymes to the weight of the disintegrated krill material isbetween 0.05% and 0.5%, preferably between 0.1% and 0.2%. Fresh caughtkrill is known for rapid and uncontrolled autolysis, or the destructionof the cells by endogenous (natural) enzymes, for which reason thetreatment described here has to be proceeded without delays when thecatch is not frozen.

The enzymatic hydrolysis also causes removing the bindings between thesoft tissue of the krill and the exoskeleton. If a mixture of enzymes isused, the mixture may also include one or more chitinases to facilitatethe further processing of the chitin-containing fractions. Chitinasesare enzymes that break down glycosidic bonds in chitin.

The enzymatic hydrolysis is finished within 100 minutes from theaddition of the endogenous enzyme(s). The preferred duration Δt of thehydrolysis is shorter, for example 45 minutes (phase 12). Relativelyshort hydrolysis duration is important, because in that case thediffusion of the fluoride from the exoskeleton particles to the othermaterial is reduced.

The hydrolysis is stopped by deactivating the hydrolytic enzyme(s),method step 13. There are many ways to deactivate the enzymes. Here itis used the thermal one: the temperature of the enzymatically processedmaterial is increased over 90° C., preferably between 92-98° C., inwhich case the hydrolytic enzymes become denatured. In practice thedeactivating of the hydrolytic enzyme(s) can be performed also during orafter the solid particle removal.

In step 14, the solid particles (e.g., krill exoskeleton) are removedfrom the enzymatically hydrolyzed and disintegrated krill material bypassage through a device based on the centrifugal force such as aconventional horizontal centrifuge and/or decanter. Although it is notnecessary to understand the mechanism of an invention, it is believedthat these solid particles, or solids, originate from the exoskeleton ofkrills and, as mentioned, contain a considerable amount of the fluoride.The decanter is operated with a force between 1,000 and 1,800 g,preferably between 1,300 and 1,500 g. Through this particle removal stepa substantial amount of fluoride, more than 90%, is removed from thekrill material. The temperature in the decanter is for example 90° C.,and if the deactivation of the enzyme(s) is done after the removal ofsolids, the temperature in the decanter is then increased to e.g. 93° C.

Next, in step 15 the hydrolyzed and disintegrated krill material withlow fluoride content is modified by passage through an extendedseparation path horizontal centrifuge (i.e., for example, a sedicanter).See, FIG. 2. In the sedicanter, the hydrolyzed and disintegrated krillmaterial, is separated into the valuable fatty portion, or PPC(Phospholipid-Peptide Complex) material fraction, and a CHF portion(Concentrated Hydrolysate Fraction).

The separation of hydrolyzed and disintegrated krill material into thePPC material is difficult because of the small density differenceswithin the krill material. The sedicanter is a modified horizontalcentrifuge including a long horizontal clarification/separation zone andgenerating high centrifugal forces (5,000 to 6,000 g). These featuresfacilitate the separation of fine, slow-settling PPC withoutemulsification. The latter is a problem in the ordinary centrifuges withshort separation zone and lower forces, and in which water is used inemptying and cleaning cycles. The dry matter concentration of PPCmaterial, pressured out from the sedicanter, is about 27-30%.

In the following step 16 the PPC material is dried to a meal to avoidthe lipid oxidation. The drying process is gentle with low temperature(0-15° C., preferably 2-8° C.) and inert conditions, which give areduced oxidative stress on the long-chain poly-unsaturated omega-3fatty acids. A lyophilisation process would also be suitable since thisavoids an over-heating of the product.

The PPC meal, or more briefly PPC, is then packed in air tight bagsunder nitrogen atmosphere for later direct use and continuation process.

A typical mass balance of the processed raw lean Antarctic krill isshown below in Table I:

TABLE I Typical Mass Balance Of Antarctic Krill From Matter 500 kg rawkrill + water Dry weight Wet PPC material 80 kg 28% PPC meal 25 kg 97%Hydrolysate 770 kg  6% CHF 78 kg 60% Fluoride-containing particles 45 kg40% Neutral oils <5 kgThe fluoride content in the hydrolyzed and disintegrated krill materialis 1.2 g/kg, whereas in the PPC it is at most 0.5 g/kg and typically 0.3g/kg. Thus, about two thirds of the fluoride has been removed.

When the PPC is further processed, components may be isolated by anextraction. In this phase, a solvent may be used (step 17 in FIG. 1).For example, to obtain krill oil from the PPC, supercritical CO₂ and/orethanol may be utililzed, either separately or in combination. Theextraction process (step 18) yields, in addition to the krill oil, aprotein hydrolysate.

Compressing and heating a material to above its critical temperature andpressure results in a supercritical fluid. The density is intermediatebetween a liquid and a gas and can be varied as a function oftemperature and pressure. Hence, the solubility of supercritical fluidscan be tuned so that selective extractions can be obtained. Due to thegas like properties, rapid extractions can be accomplished compared toliquid extractions as the diffusion rates are higher. CO₂ is a commonlyutilized supercritical fluid as its critical parameters can easily bereached. For example, one report has demonstrated a low yield of krillphospholipids by using supercritical fluid extraction at a pressure of500 bar and a temperature of 100° C. Yamaguchi (1986). A second reportprovides data on specific process conditions, which include pressure andtemperature ranges (e.g., 300 to 500 bar and 60 to 75° C.). These dataare from a pilot scale process wherein an extraction of 84 to 90% ofkrill total lipids was achieved. Bruheim et al., United States PatentApplication Publication Number 2008/0274203 (herein incorporated byreference).

Supercritical CO₂ is also non-flammable, cheap and inert, wherein suchfactors are relevant when considering industrial applicability. Theinertness results in low grade of oxidation of labile compounds duringextraction. CO₂ also has a low surface tension which is advantage sothat the extraction medium can penetrate the material efficiently. Inorder to extract more polar substances, the CO₂ can be mixed with apolar solvent such as ethanol. The level of modifier can be varied toprovide extra selectivity as well.

Consequently, currently available industrial scale supercritical fluidextraction processes using high temperatures and pressures has resultedin a low extraction efficiency of conventional krill meal therebyproviding an insufficient oil yield to provide a commercially feasiblesolution for krill extraction. Further, these currently availableextraction processes do not solve the problems discussed hereinregarding providing improved low fluoride meal and/or oil compositions.

Therefore, the improved solvent extraction methods described herein havebeen developed. In one embodiment, co-solvents are used with thesupercritical CO2, either alone or in various combinations of ethanol,hexane, acetone. For example, if ethanol is used alone as an extractionsolvent, it has been observed that krill material is less selective thanextraction with supercritical CO₂. Pronova et al., WO 2008/060163 A1. Asa result, undesirable substances are extracted into the krill oilresulting in a need for additional post-extraction clean-up/processing.Further, ethanol-only extracted krill oil tends to have higher viscosityand darker color which is independent of astaxanthin content of the oil.

In some embodiments, the present invention contemplates methods thathave unexpected findings including but not limited to: i) PPC wasextracted using low pressures (i.e., for example, between approximately177 to 300 bar) and low temperatures (i.e., for example, betweenapproximately 33 and 60° C.); and ii) high yield of lipid extract wasproduced (data available). It appears that krill meal consisting ofhydrolyzed protein allows for easier extraction of the associated lipidsin particular the phospholipid rich fraction of krill oil.

The data presented herein demonstrates that supercritical CO2 was foundto be a selective extraction method as it produced high purity extractscontaining triglycerides, phospholipids and astaxanthin with minimalbrown color and superior organoleptic quality as compared to krill oilsproduced by ethanol-only extraction and/or acetone+ethanol extraction.Brown color of krill oil is considered to be undesirable. The exactorigin of the brown color is unknown but it is believed to be associatedwith oxidation of krill lipids during the manufacture of krill mealphospholipids and/or degradation of the carotenoid astaxanthin.

The properties of such a supercritical fluid can be altered by varyingthe pressure and temperature, allowing selective component extraction.Extraction conditions for supercritical CO₂ are above the criticaltemperature of 31° C. and critical pressure of 74 bar. Addition ofmodifiers may slightly alter these values. For example, neutral lipidsand cholesterol can be extracted from egg yolk with CO₂ pressures up to370 bar and temperature up to 45° C., while using higher temperature,e.g. 55° C., would result in increased rate of phospholipid extraction.CO₂ has a high industrial applicability because it is non-flammable,cheap and inert. The inertness results in low oxidation of labilecompounds during extraction.

As mentioned, the supercritical CO₂ is fluid. Its density isintermediate between a liquid and a gas and can be varied as a functionof temperature and pressure. Hence, the solubility of supercriticalfluids can be tuned so that selective extractions can be obtained. Dueto the gas-like properties, rapid extractions can be accomplishedcompared to liquid-extractions. In the present method the extraction iseffective; even 95% of the krill oil existing in the PPC is separated.Although it is not necessary to understand the mechanism of aninvention, it is believed that the phospholipids of the feed materialare embbded in a matrix of hydrolyzed protein which means that the closeassociation between the phospholipids and hydrophobic/phosphorylatedproteins is broken thus facilitating the extraction of the lipids. Inaddition, a minimal amount of fluoride content is transferred to oilduring the CO₂ extraction process. For example, the fluoride content ofPPC is about 0.3 g/kg, but after the CO₂ extraction the fluoride contentof the krill oil is less than 0.5 mg/kg.

Alternatively, when using only supercritical CO₂ as solvent (step 19),triglycerides and/or neutral oil may be separated from the PPCcomposition subfraction. In one embodiment, supercritical CO₂-onlyextraction generates a low fluoride ‘de-oiled PPC’ composition. Althoughit is not necessary to understand the mechanism of an invention, it isbelieved that de-oiled PPC is the most valuable portion of the PPCcomposition subfraction. When thereafter, the de-oiled PPC compositionmay be extracted using ethanol as a solvent (see, step 1A), wherein aphospholipid subfraction and a protein hydrolysate fraction is alsogenerated.

In one embodiment, the present invention contemplates a systemcomprising an extraction plant, including but not limited to, a solventunit 21, vertical tank 22, separators 23 and adsorbents 24. See, FIG. 3.Normal CO₂ and possible co-solvent are fed to the solvent unit, whichcontains i.a. a pump to generate a certain pressure p and a heater togenerate a certain temperature T. The supercritical CO₂ with possibleco-solvent are then fed to the lower end of the tank 22. The feedmaterial, in this case the PPC, is fed to the tank by means of own pump.Material affected by the solvent flows out of the upper end of the tank.The separators 22 separate the extract result, for example krill oil, tooutput of the system. If ethanol is used as co-solvent, it follows theextract proper and has to be evaporated away. The CO₂ continues itscirculation to adsorbents 23, where it is cleaned, and thereafter backto the solvent unit 21.

EXPERIMENTAL Example I Production of Low Fluoride Krill Oil

5 kg batches of PPC feed material in granular form was processed usingCO₂ as solvent and azeotropic food grade ethanol as co-solvent, theweight of the ethanol being 23% of the weight of CO₂. The plant waspre-pressurised to operating pressure with CO₂ only, and ethanol wasadded when CO₂ circulation started. Solvent to feed material ratio was25:1 or greater and co-solvent to feed material ratio was 5:1. Runs werecarried out under two extraction conditions; 300 bar at 60° C., and 177bar at 40° C. See, Table II.

TABLE II Extraction conditions Run 1 Run 2 Feed Mass (g, as received)5000.5 5000.9 Extraction pressure (bar) 300 177 Extraction temperature(° C.) 60 33 First separator pressure (bar) 90 90 First separatortemperature (° C.) 41 41 Second separator pressure (bar) 48-50 48-50Second separator temperature (° C.) 39 39 CO₂ used with ethanolco-solvent (kg) 132.6 134.9 Additional CO₂ at end of run (kg) 33.1 44.5Total ethanol used (kg) 31.65 32.19The extracted material was passed through two separation vessels inseries, held at 90 bar and 45-50 bar respectively. Material collectedfrom both separators was pooled together for evaporation of ethanol.

The feed material, ‘Emerald krill meal’ granules (Olymeg or PPC), weresupplied in a sealed plastic bag containing approximately 25 kg. Thefeed material was kept frozen until used in extractions. The granuleshave a size distribution typically in the range 2 to 5 mm, but a numberof fine fragments were also present. The granules are greasy to thetouch but still break up under compression rather than smear.

Extraction results: Table III gives the mass of each extract sample fromthe two runs, and curves below the table show the cumulative extraction.A total yield of 41-42 wt % of the feed material was achieved for allruns. The runs carried out at 300 bar and 60° C. had a higher initialrate of extraction. The extraction curves indicate that the extractionis virtually complete after sample point 5 where the cumulative CO₂ usewas 21.5 to 22.0 kg of CO₂/kg feed. Estimated maximum extraction isachieved at a point where the CO₂:feed ratio is 26.5:1. See, FIG. 3(estimated maximum extraction is marked by an arrow). The ratio ofazeotropic ethanol to CO₂ was 0.24:1 for the 300 bar runs, and slightlyhigher at 0.26:1 for the lower pressure run.

TABLE III Progressive extract sample points and yields. Sample 1 2 3 4 56 Total Run 1 Cumulative CO₂ 5.5 9.1 13.4 17.8 22.0 33.1 (kg/kg feed)Extracted oil 1137 398 282 135 78 86 2115 (g, dry) Run 2 Cumulative CO₂5.6 9.1 13.5 17.5 21.5 34.4 (kg/kg feed) Extracted oil 715 496 368 220149 129 2077 (g, dry)Near complete extraction of total lipids was achieved, with averageyield of 95% of total lipids and 90% of phospholipids. The final yieldwas similar for both the high and low pressure runs, but neutral lipidswere more rapidly extracted at higher pressure. The phospholipidextraction rate was similar under both extraction conditions. Thecombined extract had an overall phospholipid level of just over 40 wt %.Phosphatidyl inositol and phosphatidyl serine were poorly extracted.

Phospholipid profiles of the lipid extracts are given in Table IV. Firstcolumn shows the phospholipid profile of the feed material (PPC or‘Olymeg®’) and the last two columns show the phospholipid profiles ofthe extracted PPC residue sampled from top and bottom of the extractioncolumn. In the lipid extracts the major phospholipid is phosphatidylcholine (PC) with proportion ranging from 72.7% to 80.4% including alkylacyl phosphatidyl choline (AAPC) and lyso forms of the PCs (LPC andLAAPC). Smaller amounts of phosphatidyl ethanolamine (PE) are present inthe feed material (5.3%) and in the lipid extracts (3.5-4.5%). Alkylacyl and lyso forms of PE (AAPE, LPE) are also present in the feedmaterial and lipid extracts. Phosphatidyl inositol (PI) and phosphatidylserine are present in the feed material (PPC), but because they arepoorly soluble in ethanol, these phospholipids are concentrated in thePPC residue.

TABLE IV Phospholipid Profiles Of Lipid Extracts (run 1) Olymeg ResidueResidue Sample 10071199 Extract 1 Extract 2 Extract 3 Extract 4 Extract5 Extract 6 (Top) (Bottom) Wt % of total PL PC 70.1 80.4 77.1 76.9 75.973.5 72.7 40.2 32.5 AAPC 8.5 8.0 9.0 9.8 9.1 10.6 9.0 7.5 7.8 PI 1.8 0.70.6 0.6 8.2 10.1 PS 1.0 5.5 8.1 LPC 6.9 4.6 5.6 5.7 6.0 6.8 7.5 13.4 8.9LAAPC 1.7 1.2 1.2 1.0 1.3 1.2 1.4 3.2 2.6 PE 5.3 3.6 4.0 3.5 3.8 3.5 4.59.4 9.4 EPLAS 0.8 0.0 0.5 0.5 0.5 0.5 0.3 1.0 2.2 AAPE 2.0 1.1 1.5 1.31.6 1.6 2.0 4.4 4.9 LPS 0.7 1.9 CL/NAPE 1.0 0.9 0.7 0.8 0.8 1.2 1.6 4.25.7 LPE 0.8 0.3 0.4 0.4 0.4 0.4 0.4 3.2 4.5 Total PL 40.88 81.46 80.96(wt % of lipid) Lipid yield 44.7 4.9 5.9 (wt %) Total PL 18.3 26.6846.03 57.94 71.34 76.13 78.50 4.0 4.8 (wt % of sample)The results presented in Table V (below) show that no free fatty acids(FFAs) are present in the lipid extracts. Sterols are present at levelsof 2% or less in the extract. Triglycerides (TAGS) were measured ataround 40 wt % and phospholipids (e.g., polar lipids) at around 50% inthe extracts. See, Table V.

TABLE V Main Components Of The Lipid Extracts Polar Total TAG lipidSterols FFA Astaxanthin lipid Run 1 40.3 46.9 1.9 ND 0.05 92.2 Run 242.1 50.2 2 ND 0.05 95.3The method and products according to the invention has been describedabove. The method can naturally vary in its details from thosepresented. The inventive idea may be applied in different ways withinthe limits set by the independent claim 1.

Example II Extraction Efficiency

This example demonstrates an exemplary analytical extraction with theSoxhlet method.

The neutral lipids are often part of large aggregates in storagetissues, from which they are relatively easily extracted. The polarlipids, on the other hand, are present as constituents of membranes,where they occur in a close association with proteins andpolysaccharides, with which they interact, and therefore are notextracted so readily. Furthermore, the phospholipids are relativelytightly bound with hydrophobic proteins and in particular with thephosphorylated proteins.

Partial hydrolysis of the protein matrix in the novel krill mealimproves the extraction efficiency of total lipid by use of organicsolvents as demonstrated by this example involving extraction of krillmeal samples with the Soxhlet method by using petroleum ether assolvent. Soxhlet method is a standard method in quantitativedetermination of fat content of foods and feeds and thus it can be usedas a reference method to determine the extractability of various krillmeals.

Extraction according to the Soxhlet method was carried out usingpetroleum ether (boiling point 30-60° C.) to evaluate the extractioncapacity using SC—CO₂. Briefly, a 10 g sample of milled krill meal wasweighed and placed in a Soxhlet apparatus and then continuouslyextracted for approximately eight (8) hours using 300 mL petroleumether. After extraction, the solvent was evaporated at 60° C. under anitrogen stream. Soxhlet F., “Die gewichtsanalytische bestimmung desmilchfettes” Dingler's Polytech. J. 232:461-465 (1879). Conventionalkrill meal was prepared as described in US 2008/0274203 (Aker BiomarineASA, Bruheim et al.) and the novel krill meal was prepared according tothe present invention.

The results show that the proportion of residual (e.g., un-extracted)lipid was twice as large in the conventional krill meal compared to thenovel krill meal. See, Table VI.

TABLE VI Extracted material Extracted lipid Residual lipid Conventionalkrill meal 79.6% 20.4% Novel krill meal 88.9% 11.1%Consequently, the extraction methods described herein have provided anunpredictable and surprising result that provides a superior productbecause of a greatly improved extraction efficiency.

Example III Determination Of Fluoride Content

This example presents one method of determining fluoride content ofkrill products as fluoride by chemical analysis using an ion selectiveelectrode.

The samples analyzed were krill meal and oil extracted from the saidkrill meal. The krill meals analyzed for fluoride content were producedby: i) a low fluoride method of present invention; and ii) a whole krillmaterial produced by a conventional process. As explained herein, themethod disclosed herein removes, in most part, the krill exoskeletonwhile the krill shell is included in the meal prepared from whole krillaccording to the conventional process. Conventional processes are, forexample, described in WO 2002/102394 (Neptune Technologies &Bioresources) and US 2008/0274203 (Aker Biomarine ASA).

The data demonstrate that by removing the exoskeleton in the process ofproducing krill meal, the fluoride content of the meal and the oilproduced from the meal have a markedly reduced fluoride content. See,Table VII.

TABLE VII Fluoride Content Comparison To Conventional Processes Processof present invention Conventional process Krill meal 300-500 ppm 1300ppm Krill oil <0.5 ppm 0.5-5 ppm

Example IV Krill Oil Color Comparison

Krill oil has typically a strong red colour arising from the carotenoidastaxanthin present in the oil at levels varying from 50 ppm to 1500ppm. Color of krill oil can be determined with a LabScan® XEspectrophotometer (Hunter Associates Laboratory, INC. Resbon, Va., USA)and reported in CIELAB colour scales (L*, a* and b* values). Deviationfrom the red colour of astaxanthin can occur when the krill biomass isprocessed at high temperature and under conditions that induceoxidation. Typical oxidation induced deviation in krill oil color is anincrease in the brownish hue. Brown color in krill oil arises fromoxidation of lipids and formation of secondary and tertiary oxidationproducts with amino residues. This process is also called non-enzymaticbrowning.

Strecker degradation products and pyrroles are products of non-enzymaticbrowning that have been characterized in samples of krill oil. Forexample, polymerization of pyrroles results in formation of brown,melatonin like macromolecules. Furthermore, pyrrole content of krill oilcan be determined spectroscopically with absorbance at 570 nm.

Samples of three krill oils will be examined for color. One produced bythe method of the present invention, one produced from frozen krill by amethod described in WO 2002/102394 (Neptune Technologies & Bioresources)and one extracted from dried krill meal with ethanol alone as describedin US 2008/0274203 (Aker Biomarine ASA). It is to be found that krilloil produced by the method of the present invention has the lowest levelof brown color determined spectrophotometrically by using CIELAB colourscales (L*, a* and b* values) and/or the lowest level of pyrrolesdetermined spectroscopically.

Example V Organoleptic Krill Oil Quality Determination

Organoleptic quality of krill oil is conventionally determined bychemical analysis of volatile nitrogenous compounds arising from thedecomposition of krill proteins and trimethyl amine oxide (TMAO).Nitrogenous compounds analyzed are total volatile nitrogen (TVN) andtrimethylamine (TMA). In simplified terms the level of nitrogenouscompounds correlate with the level of spoilage in the raw material i.e.krill biomass used for extraction of the oil.

It has become evident that, in addition to the volatile nitrogenouscompounds, a large number of volatile components with distinct odourcontribute to the sensory properties of krill oil. Many of the volatilecomponents arise from the oxidation of lipid and proteinaceous compoundsof krill biomass. Thus, a method that limits the level of oxidativedegradation in the krill biomass, will reduce the amount of volatilecomponents in krill oil.

Assessment of the organoleptic quality of different types of krill oilis to be performed by a panel of trained individuals. The sensoryproperties to be determined include several pre-defined parameters ofsmell and taste. It is to be found that the novel krill oil has animproved sensory profile compared to the other oils tested. The otheroils to be tested include one extracted from frozen krill by a methoddescribed in WO 2002/102394 (Neptune Technologies & Bioresources) andone extracted from dried krill meal with ethanol alone as described inUS 2008/0274203 (Aker Biomarine ASA).

We claim:
 1. A low fluoride krill meal composition comprising aconcentrated low fluoride krill phospholipid-peptide complex (PPC), saidPPC comprising a matrix of hydrolyzed protein and approximately 300-400g/Kg lipids and ranges between approximately 300-500 ppm fluoride,wherein the lipids comprise phospholipids and free fatty acids.
 2. Thekrill meal of claim 1, wherein said lipids comprise a range betweenapproximately 22-27% (w/w) crustacean Qmega-3 fatty acids.
 3. Acomposition comprising a low fluoride krill meal, said krill mealcomprising a concentrated low fluoride krill phospholipid-peptidecomplex (PPC), said PPC comprising a matrix of hydrolyzed protein andlipids, said fluoride level ranges from 300-500 ppm, and said lipidscomprise at least 3% (w/w) phospholipids selected from the groupconsisting of alkyl acyl phosphatidyl choline (AAPC), lyso alkyl acylphosphatidyl choline (LAAPC), alkyl acyl phosphatidyl ethanolamine(AAPE) and lyso alkyl acyl phosphatidyl ethanolamine (LPE).
 4. Thecomposition of claim 3, wherein said krill meal further comprises 20%(w/w) omega-3 fatty acids.
 5. The composition of claim 3, wherein saidLAAPC and said LPE are less than 20 wt % of total phospholipids.
 6. Thecomposition of claims 3, wherein said krill meal further comprising awater content of less than 3 wt %.